US8461124B2 - Five- and six-membered conformationally locked 2′,4′-carbocyclic ribo-thymidines for the treatment of infections and cancer - Google Patents

Five- and six-membered conformationally locked 2′,4′-carbocyclic ribo-thymidines for the treatment of infections and cancer Download PDF

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US8461124B2
US8461124B2 US12/531,394 US53139408A US8461124B2 US 8461124 B2 US8461124 B2 US 8461124B2 US 53139408 A US53139408 A US 53139408A US 8461124 B2 US8461124 B2 US 8461124B2
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aminoethylenealkoxy
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Jyoti Chattopadhyaya
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to novel carbocylic analogs of LNA and ENA compounds as disclosed in the description and drawings that follow below, and defined in the appended claims.
  • New 2′,4′-carbocyclic fused (5-/6-membered) thymidine (5-carbo-T 12a/12b, in Scheme 1, or 6-carbo-T 23 nucleosides in Scheme 2) are conformationally-constrained nucleosides (North-type).
  • the 5- or 6-carbo-T block(s) has/have been incorporated into antisense oligonucleotides (AON, See Table 3 for example) and their antisense/siRNA properties as gene-directed agent has been evaluated in order to selectively arrest translation of mRNA to protein product.
  • AON antisense oligonucleotides
  • 5- or 6-carbo-modified AONs have shown high target affinity to complementary RNA strand (T m increase of +1.5 to +5° C.
  • This study provides valuable tools regarding the optimal design of AONs or small interferring RNAs with chimeric RNAs or/and in conjunction with its 2′-modified analogs (siRNA), having completely natural phosphodiester backbone, for the therapeutic applications (down-regulation of an RNA specific to a gene or as a triplexing agent or as an aptamer) that will not only show high target affinity but also high stability towards nucleases in the blood serum.
  • siRNA 2′-modified analogs
  • Nucleobase (N) replaced by 1-Thyminyl [as in Compounds 12a, 12b and 23] or 9-Adenin
  • Antisense oligonucleotide can potentially inhibit the protein synthesis by translation arrest/steric blocking or by RNase H mediated degradation of the AON/RNA hybrid.
  • Other methods of gene silencing include formation of triplexes by base-pairing with double-helical DNA (antigene effects), or RNA interference (RNAi), by a short double-stranded RNA (siRNA).
  • RNAi RNA interference
  • siRNA short double-stranded RNA
  • phosphorothioate 7 backbone modified oligonucleotides have found some use in therapeutics.
  • Recent years have seen development of conformationally-constrained bicyclic ( FIG. 1 ) and tricyclic nucleotides, in which the sugar is locked in a definite puckered conformation.
  • Such oligonucleotides show promising properties with respect to the target RNA binding and nuclease resistance.
  • short nucleotides containing LNA 1 ( ⁇ BNA 2 ) modifications have shown unprecedented thermal stability (+3 to +8° C. per modification depending upon the sequence context).
  • the enhanced target binding property of the North-conformationally constrained bicyclic sugar units in these nucleotides has been attributed to the improved stacking between the nearest neighbors and quenching of concerted local backbone motions by LNA nucleotides in ssLNA so as to reduce the entropic penalty in the free energy of stabilization for the duplex formation with RNA.
  • These bicyclic constrained analogs have thus been extensively used to facilitate the down-regulation of genes.
  • LNA/BNA has led to the synthesis of a number of closely related analogs, in which the 2′,4′-bridge has been altered 3 or a new type of 1′,2′-bridged constraint has been introduced, such as in 1′,2′-oxetane 4 or 1′,2′-azetidine 5 analog.
  • modifications show similar or moderately depressed T m properties when compared to LNA, but the nuclease resistance or RNase H recruitment properties (for example, ENA, 6 PrNA, 7 and aza-ENA 8 ) have turned out to be relatively more favorable than those exhibited by the LNA-containing AONs.
  • the invention provides a novel synthetic strategy for the carbocyclic analogs of LNA 12 and ENA 13 thymidines (carbocyclic-LNA-T and carbocyclic-ENA-T), which have been accomplished using intramolecular free-radical ring closure reaction between a radical generated at C2′ and strategically placed double bond in the modified pentofuranose moiety of nucleoside.
  • the invention provides novel compounds as defined in claim 1 .
  • the invention provides medicaments comprising compounds according to the invention. In particular these medicaments can be used for the treatment of cancer.
  • the compounds can also be used for diagnostic and analytical purposes for identification of viral or bacteria specific DNA or RNA.
  • FIG. 1 shows structures of various bicyclic North-type conformationally-constrained ⁇ / ⁇ -D/L-pentofuranosyl nucleosides: (A) LNA 1 ; (B) amino-LNA 3 ; (C) xylo-LNA 9 ; (D) ⁇ -L-LNA 10 ; (E) ⁇ -bicyclonucleoside 11 ; (F) 1′,2′-oxetane-bridged 4 ; (G) azetidine-bridged 5 ; (H) ENA 7,11-13 , (I) aza-ENA 8 (J) PrNA 7 ; (K) unsaturated carbocyclic analog of LNA 27 14 ; (L) saturated carbocyclic analog of LNA. 14
  • FIG. 2 shows Heptenyl cyclization of 7-norborneyl radical.
  • FIG. 3 (A): 5-exo-cyclization through two transition states TS 1 and TS 2 leading to favored 5-membered carbocyclic 2′,4′-cis fused bicyclic system with R-configuration of C7′ chiral center as well as to the counterpart with the disfavored S configuration of C7′. (B): 6-exo-heptenyl cyclization through two transition states TS 3 and TS 4 leading to favored carbocyclic 2′,4′-cis fused bicyclic system with R-configuration of C8′ and its counterpart with C8′ chiral center in the disfavored S configuration.
  • FIG. 4 Autoradiograms of 20% denaturing PAGE showing degradation patterns of 5′- 32 P-labeled AONs in human blood serum (Table 1 for all AON sequences).
  • Inset A AON 1 and LNA-modified AONs 2-5.
  • Inset B Carbocyclic-LNA-modified AONs 6-9.
  • Inset C Carbocyclic-ENA-modified AONs 10-13 and
  • Inset D aza-ENA-modified AONs 14-17.
  • Time points are taken after 0, 1 ⁇ 2, 1 h, 2 h, 5 h, 7 h, 9 h, 12 h, for AONs 1-5 and 0 h, 6 h, 8 h, 12 h, 24 h, 36 h and 48 h of incubation for AONs 6-17 at 21° C. (see Experimental Section for details).
  • FIG. 5 The RNase H1 cleavage pattern of AONs 1-17/RNA heteroduplexes.
  • Vertical arrows show the RNase H cleavage sites, with the relative length of the arrow showing the extent of the cleavage.
  • the square boxes around a specific sequence shows the stretch of the RNA, which is resistant to RNase H cleavage thereby giving footprints (see PAGE autoradiograms in FIG. 6 ).
  • FIG. 6 Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5′- 32 P-labeled target RNA by E. coli RNase H1 in the AONs 1-17 after 2, 5, 10, 15, 30 and 60 min of incubation.
  • Conditions of cleavage reactions RNA (0.8 ⁇ M) and AONs (4 ⁇ M) in buffer containing 20 nM Tris-HCL (pH 8.0), 20 mM KCL, 10 mM Mgcl2, and 0.1 mM DTT at 21° C.; 0.04 U of RNase H1.
  • Inset A LNA-T modified AONs 2-5 with native AON 1.
  • Inset B carbocyclic-LNA-T modified AONs 6-9.
  • Inset C carbocyclic-ENA-T modified AONs 10-13 with native AON 1.
  • Inset D aza-ENA-T modified AONs 14-17 with native AON 1.
  • the graphs in Insets E, F, G, and H show the kinetics of RNase H1 mediated cleavage of the target RNA, the remaining fraction of target RNA is measured densitometrically and plotted as a function of time fitted to a single exponential decay function.
  • Inset E AON 1 with AONs 2-5.
  • Inset F for AON 1 with AONs 6-9.
  • Inset G AON 1 and AONs 10-13.
  • Inset H AON 1 and AONs 14-17.
  • FIG. 7 Bar plots of the observed cleavage rates of the RNase H promoted degradation of AONs 2-17/RNA heteroduplexes with various modifications (LNA-T, carbocyclic-LNA-T, carbocyclic-ENA-T and aza-ENA-T) in the AON strand at position 3, 6, 8 and 10 from the 3′-end, in comparison to that of the native counterpart AON 1.
  • the observed initial cleavage rates (sec ⁇ 1 ) of AONs 1-17/RNA heteroduplexes by E coli RNase H are found to be very similar, while in the human blood serum ( FIGS. 8-11 ) the degree of stability varied widely for the carbocyclic versus heterocyclic modified AONs reflecting their respective hydrophobic/hydrophilic properties.
  • FIG. 8 The percent of AONs 6-17 left after 48 h of incubation in the human blood serum at 21° C. Note that, under similar condition, the native (AON 1) and LNA-containing AONs (AONs 2-5) were fully degraded after 12 h, and shown in blank (red colored).
  • the human blood serum stable product (i) is (n ⁇ 1) for the AONs 6/10/14 (AONs substituted at position 3 from 3′-end) after the hydrolysis of 3′-terminal nucleotide, (ii) (n ⁇ 4) product for the AON 7/11/15 (AONs substituted at position 6 from the 3′-end), (iii) the (n ⁇ 6) product for the AONs 8/12/16 (AONs substituted at position 8 from 3′-end), whereas (iv) the (n ⁇ 8) product was formed from AONs 9/13/17 (AONs substituted at position 10 from 3′-end).
  • concentration of these final hydrolysis products formed as a result of introduction of a specific modification at a particular site was taken as 100%.
  • cleavage of one nucleotide from the 3′-end is (n ⁇ 1)
  • cleavage of two nucleotides from the 3′-end is (n ⁇ 2)
  • the ring-closure reaction involving cyclic 5-hexenyl radicals is similar to that of the open-chain systems except that the ring imposes steric constraints on the stereochemical outcome of the reaction.
  • the initial radical forms the most stable structure with bulky substituents in equatorial/pseudo-equatorial position, when the radical centre is a part of the sugar ring.
  • Ring-closure reaction occurs via attack of a radical centre oriented to pseudo-equatorial position on the axially substituted alkenyl chain resulting in the cis-fused rings.
  • the alkenyl chain preferentially occupies an axial position, since an equatorial alkenyl chain results in poor overlap of the semi-occupied molecular orbitals and ⁇ * orbitals.
  • the substituents play their role on account of their steric bulk or stereoelectronic nature as they interact with the ring atoms/substituents at different positions of alkenyl chain during intramolecular 5-hexenyl cyclization.
  • substituents on C1 and C4 atoms (5-hexenyl nomenclature) of the initial radical are the major factors dictating stereochemistry of the newly formed 1,5-bond.
  • C1 and C4 substituents are present predominantly a 1,5-cis isomer is formed.
  • C4 i.e. C4-deoxy
  • a mixture of 1,5-cis and 1,5-trans fused products is formed in the presence of vinylic oxygen the boat-like transition state is stabilized resulting in formation of 1,5-trans isomer.
  • 6-membered rings by free radical reactions involving 6-exo cyclizations of heptenyl system presents at least two problems: first, the rate constant of 6-hepetenyl cyclization is ⁇ 40 times slower than the corresponding 5-hexenyl cyclization, thus the competing radical quenching by reduction with nBu 3 SnH becomes a serious problem; second, the endo mode of cyclization is only ⁇ 7 times less rapid than the exo mode of cyclization, thus formation of the endo products also competes with the 6-membered products.
  • most of the fused bicyclic ring formation reactions studied have been of 1,2-type, i.e. the radical center is located at the neighboring carbon to the tethered double bond.
  • the 2′,4′-free-radical cyclization is a key step in our synthetic strategy to efficiently yield North-type conformationally constrained cis-fused bicyclic 5-membered and 6-membered carbocyclic analogs of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T), as it has been originally used in the construction of conformationally constrained nucleosides by Wengel's and Imanishi's group in the ionic ring-closure reaction.
  • LNA lasine-LNA-T
  • ENA carbocyclic-ENA-T
  • the synthesis starts from a known sugar precursor 1 which was selectively benzylated using a reported procedure to give the corresponding benzylated product 2.
  • the primary alcohol in sugar 2 was oxidized to the corresponding aldehyde 3 employing Swern oxidation.
  • the vinyl chain at C4 was then introduced by the Wittig reaction 6l on the crude aldehyde 3 to give the vinyl sugar 4 (87% in two steps from 2).
  • the olefin 4 was converted to C4-hydroxyethyl derivative via successive hydroboration-oxidation using 9-BBN/NaOH—H 2 O 2 to give 5 in 95% yield, which was again subjected to Swern oxidation/Wittig reaction to give the required C4-allylated sugar 6 (70% in two steps from 5) with strategically placed propenyl side chain at C4 for 5-hexenyl type free radical cyclization.
  • Compound 6 was subjected to acetolysis using a mixture of acetic anhydride, acetic acid and triflic acid to give the corresponding diacetate 7 quantitatively as ⁇ / ⁇ anomeric mixture (single spot on TLC and proven by 1 H-NMR) using.
  • the ⁇ configuration of the product 6 was confirmed by 1D differential NOE experiment, which showed 3% enhancement of H2′, and 1% enhancement of H3′ upon irradiation of H6 (d H6-2′ ⁇ 2.3 ⁇ for ⁇ anomer).
  • Deacetylation of compound 8 using 27% methanolic ammonia overnight, and subsequent esterification using phenylchlorothioformate yielded the desired precursor 10 for radical cyclization.
  • the key free radical cyclization reaction was carried out using Bu 3 SnH with radical initiator AIBN at 115° C. in degassed (N 2 ) toluene. To ensure that the radical generated has adequate lifetime to capture the double bond before it is quenched by hydrogen radical, the concentrations of Bu 3 SnH and AIBN were maintained through high dilution and slow drop-wise addition.
  • transition states TS 1 and TS 2 could be involved in the radical cyclization of the C4′-propenyl system through 5-exo-hexenyl intermediate (structure A in FIG. 3 ) in which case these transition states should represent the low-energy chair forms with newly developing C7′ methyl substituent in the pseudo-equatorial (TS 1 ) or pseudo-axial (TS 2 ) orientation.
  • the newly developing C8′ methyl substituent thus takes up the equatorial position, in which the 1,3-diaxial interaction with the C3′ axial substituent (OBn) in the newly formed fused cyclohexyl ring is absent, which makes it more favored then the product with chiral C8′ in S-configuration.
  • the orientation of the transition state, TS 4 in FIG. 3 is also in a chair conformation, with site of attack in the double-bond (C8′) and the radical centre at C3′ in the steric proximity in order to ensure 6-exo-heptenyl cyclization with minimal entropic penalty.
  • This orientation in TS 4 exhibits two 1,3-diaxial interactions, one between the 3′-O-benzyl substituent and the newly developing axial methyl substituent at C8′ and, second, between the newly forming C8′ methyl substituent and the axial proton at C6′.
  • the 1 H spectrum (FIG. S 22 in SI), showed the presence of two diastereomers, a major (12a) as well as a minor isomer (12b) in ca. 7:3 ratio.
  • the nOe enhancement ( ⁇ 12%, corresponds to ca. 2.6 ⁇ ) between H6 (thymine) and H3′ (FIGS.
  • the phosphoramidites 14 and 25 were incorporated as mono substitution in a 15 mer DNA sequence through automated synthesis on Applied Biosystems 392 RNA/DNA synthesizer for further studies.
  • the stepwise coupling yields of the modified phosphoramidite were 96% and 98%, respectively.
  • Dicyanoimidazole was used as the activating agent for 14, whereas tetrazole was used to activate 25 with 10 min coupling time for modified phosphoramidites, followed by de-protection of all base-labile protecting groups with 33% aqueous ammonia at 55° C. to give AONs 1-17 (Table 1).
  • the sequence is targeted to the coding region of the SV 40 large T antigen (TAg) and has been used in the study of antisense activity of (N)-Methanocarba-T substituted oligonucleotide and as well as in the study of antisense and nuclease stability assays of oxetane modified, azetidine modified and aza-ENA modified oligonucleotides.
  • Tg large T antigen
  • T m T m relative to RNAcompliment
  • T m * T m relative to DNA compliment.
  • T m performed with complementary DNA or RNA strand.
  • T (LNA) LNA-T 12 compound A in FIG.
  • T (5-carbo) carbocyclic-LNA-T (compound 12, Scheme 1); T (6-carbo) : carbocyclic-ENA-T (compound 23, Scheme 2); T (aza-ENA) : aza-ENA-T. 20 (compound I in FIG. 1).
  • AONs 1-17 Table 1
  • the sequence is targeted to the coding region of the SV40 large T antigen (TAg) and has been used in the study of antisense activity of (N)-Methanocarba-T substituted oligonucleotide and as well as in the study of antisense and nuclease stability assays of oxetane modified, azetidine modified and aza-ENA modified oligonucleotides.
  • AONs towards various exo and endo nucleases is necessary in order to develop any therapeutic oligonucleotides (antisense, RNAi, microRNA or triplexing agents).
  • the first generation nuclease resistant antisense phosphorothioates were followed by 2′-O-alkylated modifications.
  • Recent conformationally-constrained molecules LNA, ENA, bicyclic, and tricyclic, aza-ENA, oxetane, azetidine etc.
  • LNA, ENA, bicyclic, and tricyclic, aza-ENA, oxetane, azetidine etc. have also shown enhanced nuclease stability as compared to the natural deoxy counterpart.
  • Egli et al. have demonstrated that charge effects and hydration properties are important factors in influencing the nuclease stability of AONs with normal phosphodiester backbone.
  • the modified AON 14 with T (aza-ENA) at position 3 from 3′-end showed full hydrolysis of the 3′-terminal nucleotide within 5 h to give the AON fragments with n ⁇ 1 (ca. 85%) and n ⁇ 2 sequence (ca. 15%), which were further hydrolyzed to ca. 65% and 35% respectively after 12 h. No further cleavage had been observed until 48 h (Inset D in FIG. 4 and plots of percent AONs remaining as a function of time in FIG. S 78 in SI). This means that AON 14 with n ⁇ 1 nucleotide sequence was being hydrolyzed steadily to give the AON with n ⁇ 2 nucleotide sequence.
  • FIG. S 76 in SI AONs 11-13 (6-membered carbocyclic modifications at position 6, 8 and 10 from 3′-end giving n ⁇ 4, n ⁇ 6 and n ⁇ 8 fragments, respectively, FIG. S 77 in SI) and AONs 15-17 (6-membered aza-ENA modifications at position 6, 8 and 10 from 3′-end giving n ⁇ 4/n ⁇ 5, n ⁇ 6/n ⁇ 7 and n ⁇ 8/n ⁇ 9 fragments, respectively.
  • FIG. S 78 in SI AONs 11-13 (6-membered carbocyclic modifications at position 6, 8 and 10 from 3′-end giving n ⁇ 4, n ⁇ 6 and n ⁇ 8 fragments, respectively.
  • FIG. S 77 in SI AONs 15-17 (6-membered aza-ENA modifications at position 6, 8 and 10 from 3′-end giving n ⁇ 4/n ⁇ 5, n ⁇ 6/n ⁇ 7 and n ⁇ 8/n ⁇ 9 fragments, respectively.
  • FIG. S 78 in SI AONs 11-13
  • the 3′-terminal nucleotide is hydrolyzed by 3′-exonuclease in AON substituted by the 5- or 6-membered carbocyclic residue at position 3 (AONs 6 and 10) to give only the n ⁇ 1 fragment.
  • AONs with single substitution at either position 6 (AONs 7 and 11) or 8 (AONs 8 and 12) or 10 (AONs 9 and 13) similarly gives only n ⁇ 4, n ⁇ 6 or n ⁇ 8 fragments, respectively.
  • the residual AON sequences remained at the 5′-end of the modification site were found to be intact for more than 48 h in the human blood serum (see PAGE autoradiograms in Insets: B-C in FIG.
  • the 2′- or 6′-alkoxy substituted carbocyclic nucleotide units (three units at the 3′-end) in the modified AON enhanced stability of AON on fetal calf serum from 2.5 times for [6′ ⁇ -carbocyclic-2′-deoxy]-T substitution to 24 times for [6′ ⁇ -carbocyclic-2′-O—(CH 2 ) 4 —NH 2 or 2′-O—(CH 2 ) 3 -Ph]-T substitution), compared to that of the native.
  • replacement of substituents involved in natural enzyme-substrate complex results in poor recognition and processing by the nucleolytic enzymes, thereby resulting in the nuclease stability.
  • nuclease stability was enhanced when 2′-bulky substituent was introduced in the carbocyclic nucleosides Subsequently, it was proved that the native ribonucleoside with 2′-O-alkyl substituent either by its bulk or by its stereoelectronic modulation of the hydration can bring about nucleolytic stability.
  • the AONs containing 5-membered carbocyclic-LNA versus AONs with the 6-membered carbocyclic-ENA show enhanced, but identical, blood serum stability, thereby showing that the steric bulk is relatively unimportant. This is in sharp contrast to the conclusion drawn by comparison of the LNA versus ENA modified AONs, in that the latter is 2.5-3 times more stable than the former, 13 apparently, according to the authors, owing to an extra methylene linker.
  • the 3′-exonuclease (SVPDE) stability had also been observed to be higher for the 2′-O-GE and 2′-O-aminopropyl modifications, as well as for the 4′- ⁇ -C-aminoalkylthymidine AONs, which showed complete nuclease resistant upon incorporation of five modified nucleotides as mixmers, compared to the native counterpart.
  • nuclease digestion studies involving 4′- ⁇ -C-aminoalkylthymidine AONs showed that longer chain alkyls were less potent in providing stability against nuclease, and hinted at the role of ammonium ions in providing the stability 30 .
  • Table S5 in SI thus shows that the energy of stabilization of the solvated of 5-membered (12a and 12b) and 6-membered (23) carbocyclic nucleosides compared to their oxygen and nitrogen containing counterparts decreases in the following order: 6-membered carbocyclic-ENA-T (12.2 kcal mol ⁇ 1 )>.
  • 6-membered carbocyclic-ENA-T (12.2 kcal mol ⁇ 1 )>.
  • 5-membered carbocyclic-LNA-T (12.9 kcal mol ⁇ 1 )>aza-ENA-T (15.2 kcal mol ⁇ 1 )>ENA-T (15.6 kcal mol ⁇ 1 )>LNA-T (16.8 kcal mol ⁇ 1 ).
  • RNA complementary to AONs (AONs 1-17 in Table 1) formed duplexes and were found to be good substrates for RNase H but with varying cleavage sites ( FIG. 5 ) depending on the site of modification in the AON strand as shown in PAGE autoradiograms (Insets A-D in FIG. 6 ).
  • the main observations are as follows: (i) The RNA compliment of the unmodified native AON 1/RNA duplex was cleaved quite randomly with a slight preference after A-8 position.
  • RNase H does not recognize the hydrophobic (as in carbocyclic-LNA, carbocyclic-ENA) or hydrophilic (LNA and aza-ENA) character of the substituent at the 2′-position of the modified nucleosides, but interrogates only at the subtle difference in the flexibility of the North-type sugar puckering (2′,4′ versus 1′,2′) in that we observed different cleavage sites and footprint pattern (4-5 nt gaps) for isosequencial 1′,2′-constrained North-East sugar puckered oxetane and azetidine modified AONs compared to those of the more rigid 2′,4′-constrained systems (LNA, carbocyclic-LNA, carbocyclic-ENA and aza-ENA modified AONs).
  • the cleavage rates of RNase H digestion were determined by densitometric quantification of gels and subsequently by plotting the uncleaved RNA fraction as a function of time (Insets E-H in FIG. 6 ). The reaction rates were obtained by fitting the degradation curves to single-exponential decay functions. The relative cleavage rates with carbocyclic-LNA, carbocyclic-ENA, aza-ENA and LNA modified AON/RNA duplexes were found to be very similar to that of the native counterpart irrespective of the type and the site of modification in the AON strand (compare the relative rates in FIG. 14 ).
  • FIGS. S 76 -S 78 in SI may produce the highly desired pharmacokinetic properties because of their stability, and consequently a net reduction of the required dosage.
  • the modified AONs with all thymidines replaced with the conformationally-constrained (North- or South-locked) 2′-deoxy-methanocarba-nucleoside or LNAs or tricyclo-DNA did not recruit RNase H.
  • carbocyclic-LNA/-ENA-T into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) [compared to those of the native (fully degraded ⁇ 3 h) and the LNA-modified AONs (fully degraded ⁇ 9 h) and aza-ENA ( ⁇ 85% stable in 48 h)].
  • This enhanced stability of the carbocyclic-LNA/-ENA-T containing AONs however do not compromise the recruitment of the RNase H to cleave the complementary RNA in the modified AON/RNA heteroduplex, compared to that of the native.
  • This enhanced life-time of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties and consequently a reduction of the required dosage and the toxicity while down-regulating a message in vivo, which may make the carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense or RNAi therapeutic agent.
  • Oxalyl chloride (10.7 mL, 125 mmol) was added to dichloromethane (350 mL) cooled at ⁇ 78° C. DMSO (15 mL, 200 mmol was added dropwise to this solution in about 30 min. After stirring for 20 more min a solution of 2 (20 g, 50 mmol) in dichloromethane (100 mL) was added dropwise to this mixture in about 20 min and left to stir at ⁇ 78° C. for another 30 min. DIPEA (60 mL, 350 mmol) was added to this cooled mixture and allowed to warm to room temperature. Water was added to the reaction and twice extracted with dichloromethane (100 mL).
  • Oxalyl chloride (6.2 mL, 72.46 mmol) was added to dichloromethane (200 mL) cooled at ⁇ 78° C.
  • DMSO 11 mL, 145 mmol was added dropwise to this solution in about 30 min After stirring for 20 more min a solution of 5 (15 g, 36.23 mmol) in dichloromethane (100 mL) was added dropwise to this mixture in about 20 min and left to stir at ⁇ 78° C. for another 45 min.
  • DIPEA 35 mL, 200 mmol was added to this cooled mixture and allowed to warm to room temperature. Water was next added to the reaction and twice extracted with dichloromethane (100 mL).
  • Acetic anhydride (17 mL, 175 mmoles) and acetic acid (87 mL) were added to 4 (6.0 g, 14 mmoles) and cooled, triflic acid (0.1 mL, 0.7 mmoles) was added to it and stirred. After 30 min the reaction was quenched with cold saturated NaHCO 3 solution and extracted with dichloromethane. The organic layer dried and evaporated. The crude was co evaporated with dry CH 3 CN thrice and dissolved in the same. Thymine (2.4 g, 19 mmol) and N,O-bis(trimethylsilyl)acetamide (9.6 mL, 38 mmol) was added to this solution and refluxed for 45 min till suspension becomes a clear solution.
  • the four signals in 31 P NMR have been integrated and are found to be in the ratio of 7:3 as found in the 1 H NMR spectrum of 12a/12b.
  • the integration is included in the SI (Inset in FIG. S 42 ).
  • Oxalyl chloride (2.48 mL, 29.20 mmol) was added to dichloromethane (100 mL) cooled at ⁇ 78° C.
  • DMSO 3.3 mL, 46.72 mmol
  • DIPEA 13 mL, 100 mmol
  • Acetic anhydride (9.5 mL, 50 mmol) and acetic acid (50 mL) was added to 4 (3.5 g, 8.2 mmol) and cooled on ice bath, triflic acid (0.03 mL, 0.4 mmol) was added to it and stirred. After 30 min the reaction was quenched with cold saturated NaHCO 3 solution and extracted with dichloromethane. The organic layer dried and evaporated. The crude was co-evaporated with dry CH 3 CN thrice and dissolved in the same.
  • Thymine (1.5 g, 12.37 mmol) and N,O-bis(trimethylsilyl)acetamide (5.0 mL, 24.75 mmol) were added to this solution and refluxed for 45 min till suspension becomes a clear solution.
  • This solution was cooled to 0° C. and TMSOTf (1.71 mL, 9.9 mmol) was added dropwise and left to stir overnight.
  • the reaction was quenched with saturated NH 4 Cl solution and extracted with dichloromethane. The organic layer was dried, evaporated and treated with 27% methanolic ammonia overnight.
  • the nucleoside 20 (2.8 g, 5.7 mmol) was evaporated thrice with dry pyridine and dissolved in the same. To this pre-cooled solution was added DMAP (0.69 g, 5.7 mmol) and then dropwise was added phenyl chlorothionoformate (1.15 mL, 8.55 mmol) and reaction stirred overnight. Reaction was quenched with saturated solution of NaHCO 3 and extracted with dichloromethane. Organic layer was dried over MgSO 4 concentrated and chromatographed over silica gel (10-30% ethyl acetate in cyclohexane, v/v) to give 21 as yellowish foam (2.1 g, 3.42 mmol, 60%).
  • samples (1 ⁇ M of AON and 1 ⁇ M RNA mixture) were pre-annealed by heating to 90° C. for 5 min followed by slow cooling to 4° C. and 30 min equilibration at this temperature and are average of at least three independent runs.
  • oligoribonucleotide, oligodeoxyribonucleotides were 5′-end labeled with 32 P using T4 polynucleotide kinase, [ ⁇ - 32 P]ATP and standard protocol.
  • Labeled AONs and the target RNA were purified by 20% denaturing PAGE and specific activities were measured using Beckman LS 3801 counter.
  • AONs (6 ⁇ L) at 2 ⁇ M concentration (5′-end 32 P labeled with specific activity 80 000 cpm) were incubated in 26 ⁇ L of human blood serum (male AB) at 21° C. (total reaction volume was 36 ⁇ L) and the experiments were repeated twice up to 48 h. Aliquots (3 ⁇ L) were taken at 0, 30 min, 1, 2, 5, 7, 8, 9, 12, 24, 36, 48 h and quenched with 7 ⁇ L of solution containing 8 M urea and 50 mM EDTA, resolved in 20% polyacrylamide denaturing (7 M urea) gel electrophoresis and visualized by autoradiography.
  • the geometry optimizations of the modified nucleosides have been carried out by GAUSSIAN 98 program package at the Hartree-Fock level using 6-31G** basis set.
  • the atomic charges and optimized geometries of compounds 12a, 12b, and 23 were then used as AMBER 87 force field parameters employed in the MD simulations.
  • the protocol of the MD simulations is based on Cheathan-Kollman's 88 procedure employing modified version of Amber 1994 force field as it is implemented in AMBER 7 program package.
  • Malgorzata Wenska was responsible for the scale up of compound 23 using PS's procedure. Jharna Barman has performed enzymological experiments with PS. Wimal Pathmasiri has performed detailed NMR characterization by 500/600 MHz NMR and simulation. Oleksandr Plashkevych has performed molecular structure analysis based on the NMR experiments and ab initio and MD simulations.

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US10538764B2 (en) 2014-06-16 2020-01-21 University Of Southampton Reducing intron retention
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US10941405B2 (en) 2015-10-09 2021-03-09 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
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